Science Collected Works - Theses
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ItemDissecting apicoplast targeting in plasmodium falciparum and toxoplasma gondii(University of Melbourne, 2004)The phylum Apicomplexa is a group of obligate intracellular parasites, responsible for a wide range of diseases of humans and livestock. Two notable species are Toxoplasma gondii and Plasmodium falciparum. T. gondii is the causative agent of toxoplasmosis and causes congenital birth defects and medical complications in AIDS patients. Plasmodium falciparum, on the other hand, is responsible for the most deadly of all human diseases � cerebral malaria. The plastid (termed apicoplast) of apicomplexan parasites contains many plant-like features and is indispensable. It therefore provides a new avenue of treatment for tackling the huge medical and economic burden this group of parasites cause. Like other plastids the apicoplast contains its own genome, but only encodes a fraction of the proteins that it requires to meet all its biological functions. Instead, most of the proteins that function within this organelle are encoded in the nucleus and are post-translationally targeted across the apicoplast�s four membranes. In chapter one I review the field of plastid targeting, including the targeting across two, three and four membrane plastids. I then extensively review data on the rapidly expanding field of apicoplast protein targeting. The study of protein targeting has been greatly enhanced by the ability to genetically modify organisms and P. falciparum is no exception. However, creating transgenic P. falciparum is challenging due to the parasites low transfection efficiency and the troubles associated with creating the transfection vectors. In chapter two I describe two new series of P. falciparum transfection vectors based around Gateway� recombinatorial cloning that I have used to create the transfection vectors described throughout this thesis. These new plasmids are high copy number in E. coli and circumvent many of the problems associated with making P. falciparum transfection vectors to express fluorescent protein chimeras. Indeed, the second set of vectors based on Gateway�s multisite cloning system completely eliminates the need for restriction enzyme mediated cloning methods. In chapter two I also describe a new parasite fixation method for light microscopy and together with the new vectors I show their application in the study of P. falciparum cell biology. By creating different transgenic cell lines I perform localization studies on several important organellar proteins and also investigate the morphology of the endoplasmic reticulum (ER) and mitochondrion throughout the intraerythrocytic lifecycle. By creating double transgenic parasites expressing two fluorescent proteins I also preliminarily characterize the relationship between the apicoplast and mitochondria throughout the intraerythrocytic lifecycle. Targeting nuclear encoded proteins across the apicoplast�s four membranes requires a bipartite N-terminal extension. The first domain resembles a eukaryotic signal peptide, which is then followed by a domain resembling a plant transit peptide. In chapter three I investigate the nature of the first targeting domain - the apicoplast signal peptide. By swapping signal peptides with other secretory proteins, I show that apicoplast signal peptides contain no novel information and show that apicoplast protein targeting initiates via the general secretory pathway. I also show that the apicoplast shares an intimate relationship with the ER and show, using brefeldin A as an effector molecule, that nuclear-encoded apicoplast-targeted proteins, most likely do not pass through the Golgi apparatus. In chapter four I investigate the properties of the second targeting domain � the apicoplast transit peptide. Like plant plastid transit peptides, apicoplast transit peptides are enriched in hydrophilic and basic amino acids and depleted in acidic residues. Other lab members used these characteristics to create an algorithm to predict apicoplast proteins (termed �PlasmoAP�). I demonstrate the biological importance of rules embedded within this algorithm by creating a series of point mutations in model apicoplast transit peptides in both P. falciparum and T. gondii. I also show that the exact position of N-terminal positive charge is not important but that N-terminal transit peptide positive charge is more important than C-terminal positive charge. This set of experiments agrees strongly with the rules embedded in PlasmoAP and provides great confidence in this algorithm�s prediction of the apicoplast proteome. I also show using a point mutagenesis approach, that binding of the chaperone Hsp70 is most likely important in apicoplast transit peptide fidelity and that phenylalanine at +1 (relative to the signal peptide cleavage site) and hydroxylated amino acids may also be important apicoplast transit peptide features. In chapter five I further my analysis of apicoplast transit peptides. I demonstrate the evolutionary conservation of the apicoplast transit peptide by showing the functional equivalency of three plant transit peptides. Furthermore, with aid of PlasmoAP I show that both randomly generated sequence and exons from non-apicoplast genes can behave like transit peptides in vivo, illustrating that apicoplast transit peptides are just a simple collection of amino acids with certain properties. This also suggests that these loose parameters of transit peptides would allow these targeting signals to be easily acquired throughout the process of organelle evolution. In summary, this thesis dissects the mechanisms and pathway adopted by apicomplexan parasites to target proteins to the apicoplast. The work presented in this thesis outlines the biological parameters governing apicoplast targeting, and taken in a broader sense answers many questions relating to targeting to plastids with four membranes. This is also the first study to illustrate the simplicity of plastid transit peptides. This work has also led to the robust prediction of the apicoplast proteome, which in turn can be used as an avenue to explore new antiparasitic drugs.